Product Overview

Cap1 capping system contains Vaccinia Capping Enzyme, mRNA Cap 2'-O-Methyltransferase, and other capping-related components, which can incorporate Cap 0 or Cap 1 structures at the 5' end of uncapped mRNAs.

This structure is identical to the natural cap structure in eukaryotic organisms, which can significantly reduce the immunogenicity of exogenous mRNAs, enhance RNA stability, and improve translation efficiency.

The Vaccinia Capping Enzyme in this kit is derived from recombinant E. coli carrying cowpox virus MR gene. The enzyme is composed of D1R and D12L double subunits, and possesses three enzymatic activities: RNA triphosphatase activity, Guanylyltransferase activity, and N7-methyltransferase activity.

The D1 subunit carries out RNA triphosphatase and Guanylyltransferase functions; the D12 subunit acts as N7-methyltransferase to specifically attach the 7-methylguanosine Cap 0 structure to the RNA 5' terminus.

mRNA Cap 2'-O-methyltransferase is expressed from recombinant E. coli containing the cowpox virus methyltransferase gene. The enzyme uses S-adenosylmethionine (SAM) as methyl donor to methylate Cap 0 RNA and generate Cap 1 structure — a methyl group added to the 2'-O position of the first nucleotide next to the 5' cap.

The Cap1 capping system catalyzes four sequential enzymatic reactions (Figure 1):

(i) RNA triphosphatase: Cleaves RNA 5'-triphosphate into diphosphate.

(ii) Guanylyltransferase: Links GTP to the RNA 5'-diphosphate of the first nucleotide.

(iii) N7-methyltransferase: Mediates N7 methylation of guanine with SAM as cofactor.

(iv) mRNA Cap 2'-O-methyltransferase: Methylates the 2'-O site of the first nucleotide adjacent to the Cap 0 cap.

This kit can cap 50 μg RNA per single reaction and the reaction can be scaled up to prepare milligram-level RNA.

Capped mRNA shows boosted translational activity, which markedly elevates protein expression in transfection and microinjection assays.

Figure 1. mRNA enzymatic capping pathway involving Vaccinia Capping Enzyme and mRNA Cap2'-O-methyltransferase.

Key Features

High Capping Efficiency

Achieves >95% capping efficiency for standard RNA sequences, supporting the preparation of high-quality capped mRNA.

Enzymatic Generation of Cap 0 and Cap 1 Structures

Combines Vaccinia Capping Enzyme and mRNA Cap 2′-O-Methyltransferase to convert uncapped RNA into Cap 0 or Cap 1 mRNA through sequential enzymatic reactions.

Pre-Denaturation-Free Workflow

Compatible with workflows that do not require RNA pre-denaturation, simplifying experimental procedures and facilitating scale-up.

Scalable mRNA Processing

Processes up to 50 μg of RNA per standard reaction, with reactions readily scalable to support milligram-level mRNA preparation.

Complete Capping Solution

Supplied with the enzymes and reagents required for post-transcriptional capping, providing a convenient and standardized workflow.

Applications

Post-Transcriptional Cap 1 mRNA Preparation

Suitable for the conversion of uncapped RNA transcripts into Cap 0 or Cap 1 mRNA for applications requiring eukaryotic-like cap structures.

mRNA Research and Preclinical Studies

Applicable to a range of mRNA applications, including in vitro translation, transfection, microinjection, and preclinical mRNA studies.

mRNA Process Development and Manufacturing

Can be integrated into enzymatic mRNA production workflows for process development and scaled RNA preparation.

Sequential Workflow with IVT Systems

Designed for use as a downstream capping step following in vitro transcription, and can be used in conjunction with the High-Yield T7 In Vitro Transcription Kit to establish a complete mRNA synthesis workflow.

Performance Data

Component List

FAQ

Can this system generate both Cap 0 and Cap 1 structures?

Yes. The system can be used to produce either Cap 0 or Cap 1 structures from uncapped RNA through sequential enzymatic reactions.

What type of RNA can be used as the input?

The system is designed for use with uncapped RNA transcripts, including RNA generated by in vitro transcription workflows.

What capping efficiency can be achieved?

For standard RNA sequences, the system can achieve capping efficiencies exceeding 95% under recommended reaction conditions.

Can this system be used for large-scale mRNA preparation?

Yes. Each standard reaction can process up to 50 μg of RNA, and the workflow can be readily scaled up to support milligram-level mRNA production.

Does this system need to be used with a specific IVT kit?

No. The system can be used with uncapped RNA generated from various IVT workflows. It can also be combined with the High-Yield T7 In Vitro Transcription Kit to establish a complete mRNA synthesis workflow.

How should the kit be stored and transported?

The kit should be transported below 0°C and stored at −25 to −15°C to maintain optimal stability and performance.